OBJECTIVEThe current treatment for advanced head and neck squamous cell carcinoma continues to result in poor outcomes. The purpose of this study is to investigate the benefit of fibroblast growth factor 2‐targeted adenovirus‐mediated mutant‐Rad50 (FGF2‐Ad‐Rad50) gene transfer in enhancing chemosensitization for head and neck squamous cell carcinoma and reducing chemotoxicity.STUDY DESIGNRandomized controlled laboratory study.SETTINGUniversity of Pennsylvania, Philadelphia, PA.SUBJECTS AND METHODSHuman head and neck squamous cell carcinoma tumor cells and a mouse model with human head and neck squamous cell carcinoma were used for this study. There were five mice in each study group. FGF2‐fab' molecule was conjugated with an adenoviral mutant‐Rad50 construct. FGF2‐targeted transgene expression efficiency was evaluated in vitro. Tumor cytotoxicity and growth inhibition were examined after combined FGF2‐Ad‐Rad50 with cisplatin treatment in vitro and in vivo. Anti‐tumor mechanisms were investigated.RESULTSFGF2‐targeted gene transfer approach significantly improved transgene expression in head and neck squamous cell carcinoma tumor cells over a nontargeted approach (207.51 ± 33.62 vs 51.44 ± 8.28, respectively). FGF2‐Ad‐Rad50 with cisplatin demonstrated a superior tumor suppression effect (264.5 ± 124.1 mm3 vs 567.1 ± 267.6 mm3), increased DNA double‐strand breaks (1349 ± 51.67 vs 774 ± 28.56), and anti‐angiogenesis (%ROI: 0.76% ± 0.38% vs 2.10% ± 1.66%) in tumor cells over nontargeted adenovirus.CONCLUSIONCombination of FGF2‐Ad‐Rad50 with cisplatin significantly improves anti‐tumor effect by targeting DNA repair systems and tumor angiogenesis. The great benefit of this strategy supports clinical trial for novel treatment of head and neck squamous cell carcinoma.